Introduction

Heart failure is a common cardiovascular disease with poor prognosis that develops when the heart is unable to pump blood and maintain tissue perfusion (Fang et al., 2008; Yancy et al., 2013). Despite improvements in the treatment of cardiovascular diseases, such as coronary heart disease and hypertension, the prognosis of heart failure remains poor (Braunwald, 2013). Several mechanisms contribute to the development of heart failure following valve disease, cardiomyopathy, or after myocardial infarction (Frey and Olson, 2003; Jessup and Brozena, 2003; Heineke and Molkentin, 2006). In most cases, cardiac remodeling occurs in response to environmental demands, and various stimuli, such as hormonal activation and hypertension, inducing cardiac hypertrophy. Hypertrophic growth is the primary mechanism to reduce stress on the ventricular wall; however, the heart undergoes irreversible decompensation with prolonged stress, resulting in heart failure (Hill and Olson, 2008).

All cells possess transmembrane signaling systems responsive to extracellular stimuli. G protein–coupled receptors (GPCRs) are the largest superfamily of cell surface receptors and are involved in numerous physiological and pathological processes (Katritch et al., 2013). GPCR-mediated signaling is implicated in various diseases, including metabolic, immunological, and neurodegenerative disorders, as well as cancer and infection (Heng et al., 2013); thus, GPCRs are considered to be attractive drug targets (Overington et al., 2006).

In the heart, GPCRs regulate cardiac function in response to extracellular stimuli, such as catecholamines and angiotensin II, and play a role in cardiac dysfunction and fibrosis (Wettschureck and Offermanns, 2005). GPCR inhibitors are widely used to treat patients with heart failure (Kang et al., 2007; Capote et al., 2015). Although the heart expresses several GPCRs (Regard et al., 2008), only β adrenergic and angiotensin II receptors antagonists are clinically used as a long-term treatment for patients with chronic heart failure. Despite these available therapies, mortality and hospitalization rates have remained relatively high for over a decade, suggesting that additional uncharacterized factors may also mediate disease pathophysiology (Tamargo and López-Sendón, 2011).

Results and discussion

Continuous Crhr2 activation causes heart failure in mice

A systematical search was performed to identify GPCRs expressed in cardiomyocytes and related to heart failure. For this, we performed non-biased quantitative RT-PCR (qRT-PCR) analysis to determine the gene copy number of 475 GPCRs in adult murine cardiomyocytes 2 wk after sham procedure or transverse aortic constriction (TAC; Fig. S1). Data revealed that adult murine cardiomyocytes expressed about 80 GPCRs (>5 copies per ng of RNA), the most abundant being Crhr2, Adrb1, Ptger1, and Gpr157 (Fig. 1 A). Crhr2 expression was markedly increased at the gene and protein level in the left ventricle 2 wk after TAC, whereas that of Adrb1 was decreased and Ptger1 expression was unchanged (Fig. 1, B and C). Moreover, TAC significantly increased Ucn2 levels in the blood (Fig. 1 D). Western blot analysis of various human tissues indicated that Crhr2 is exclusively expressed in the heart and is undetectable in other tissues (Fig. 1 E). Together, these results indicate that Crhr2 is highly expressed in cardiomyocytes and increases after pressure overload–induced heart failure.

To examine the functional significance of Crhr2 in cardiomyocytes in vivo, we generated mice with tamoxifen-inducible cardiomyocyte-specific Crhr2 deficiency (cmc-Crhr2 KO) by mating αMHC-Cre-ERT2 mice with Crhr2flox/flox animals. The efficiency of tamoxifen-inducible recombination was analyzed by Western blotting, which showed that Crhr2 was undetectable in cardiomyocytes isolated from tamoxifen-treated cmc-Crhr2 KO mice (Fig. 2 A). No differences were observed in the ratio of left ventricular weight to tibia length or cardiac function before and after tamoxifen treatment in cmc-Crhr2 KO mice (Fig. 2, B and C). Notably, cmc-Crhr2 KO mice failed to show a Ucn2-induced hypertrophic response (Fig. 2 B). In addition, although 4 wk of continuous Ucn2 infusion decreased fractional shortening in control mice, this was significantly attenuated in cmc-Crhr2-KO mice (Fig. 2 C). These results indicate that continuous Ucn2 infusion directly affects cardiomyocytes via Crhr2.

Data from the present study shows that Crhr2 is abundantly expressed in cardiomyocytes and its expression increases in pressure overload–induced cardiac dysfunction. There are two Crhr isoforms, termed Crhr1 and Crhr2 (Bale and Vale, 2004). Crhr1 is mainly expressed in the brain and pituitary gland, whereas Crhr2 mRNA expression in mice is found in peripheral tissues, such as the heart, liver, and skeletal muscle (Kishimoto et al., 1995; Stenzel et al., 1995; Chen et al., 2005). Moreover, Crhr1 appears to be responsible for stimulating the release of adrenocorticotrophic hormone in stress responsivity; however, the role of endogenous Crhr2 remains unclear. Our study demonstrated that Crhr2 protein is abundantly expressed in the human heart, and plasma Ucn2 levels were higher in patients with heart failure compared to healthy controls, indicating that endogenous Crhr2 is associated with heart failure in humans. Our findings are supported by clinical trials in heart failure that reported increased plasma Ucn2 was associated with heart failure (Davis et al., 2007a,b; Adão et al., 2015).

Because estrogen increases Ucn2 levels and Crhr2 expression in cardiomyocytes (Cong et al., 2014), another consideration is whether there are gender differences in the role of Ucn2 in the heart. We implanted female mice with osmotic pumps that release Ucn2 and found that 4 wk of Ucn2 infusion caused a significantly increased left ventricular weight to tibia length and decreased ventricular fractional shortening (unpublished data). Additionally, cmc-Crhr2 KO female mice were resistant to TAC-induced cardiac hypertrophy and Crhr2 antagonist treatment protected female mice from further TAC-induced cardiac dysfunction similar to that observed in male mice (personal correspondence). These findings indicate that there are no gender differences in the role of Crhr2 in heart failure.

Based on our findings, although acute Ucn2 injection has been shown to increase cardiac function, chronic elevations in Ucn2 are associated with cardiac dysfunction. These opposing effects may be similar to that found with cardiac β adrenergic receptors, which are also coupled to Gαs. Although adrenaline treatment is beneficial during the acute phase, β blockers have become a frontline drug for the treatment of chronic heart failure (Braunwald, 2001). It is known that cellular signaling is adaptive in acute heart failure, and can cause maladaptive cardiac remodeling over prolonged periods of time. Most GPCRs are coupled to more than one heterotrimeric G-protein, resulting in activation of specific intracellular pathways. Cardiac Gαs transgenic mice exhibit progressive cardiac dysfunction with cardiac apoptosis, cardiac hypertrophy, and cardiac fibrosis as the mice age (Iwase et al., 1996; Geng et al., 1999). Cardiac PKA transgenic mice display ventricular dilation and decreased cardiac function (Antos et al., 2001), and PKA phosphorylation is significantly elevated in failing hearts from humans and animal models (Marx et al., 2000). Similarly, CREB phosphorylation promotes target gene expression (Mayr and Montminy, 2001; Altarejos and Montminy, 2011), although the functional significance of CREB in the normal heart is controversial. For instance, cardiac-specific overexpression of dominant-negative CREB induced cardiac dysfunction (Fentzke et al., 1998); however, CREB-deficient mice display no changes in cardiac morphology (Matus et al., 2007). Nevertheless, mechanistically, CREB has been implicated in heart failure, and CREB Ser133 phosphorylation enhances the expression of genes related to heart failure (Guo et al., 2010; Chien et al., 2015).

Although activation of AKT initially promotes physiological growth of the heart, constitutive AKT activation leads to pathological cardiac hypertrophy and dysfunction (Chaanine and Hajjar, 2011; Shimizu and Minamino, 2016). In a murine pressure overload model, heterozygous AKT1-deficient (Akt1+/-) mice are resistant to pressure overload–induced cardiac dysfunction (Shimizu et al., 2010); however, the mechanism by which this occurs requires further investigation.

We show in this study that plasma Ucn2 levels significantly increase in patients with heart failure and in mice with pressure overload–induced cardiac dysfunction. Although Ucn2 mRNA is detected in various tissues, including the brain and heart (Hillhouse and Grammatopoulos, 2006), the secretory organ of Ucn2 remains unclear. In a mouse model of myocardial infarction, Ucn2 expression increases in cardiomyocytes (Li et al., 2013), leading to the hypothesis that cardiomyocytes secrete Ucn2 in heart failure. We measured plasma Ucn2 levels in the peripheral arterial, venous, and coronary sinus blood from patients with NIDCM, but found no significant increase in the coronary sinus sample (unpublished data). Thus, further studies are required to pinpoint the cellular origin of Ucn2 in the context of heart failure.

In conclusion, our data show that Crhr2 activation plays a critical role in the development of heart failure and that Crhr2 antagonist treatment prevents cardiac dysfunction in model mice. Clinical analysis showed significantly elevated plasma Ucn2 in patients with NIDCM compared to healthy subjects. Therefore, Ucn2 may serve as a prognostic indicator for response to Crhr2 inhibitor treatment and facilitate the development of more effective therapies for chronic heart failure.

mRNA expression analysis

To analyze GPCR expression, RNA was extracted from adult mouse cardiomyocytes with an RNeasy Mini kit (QIAGEN) according to the manufacturer’s protocol and reverse-transcribed with a QuantiTect Reverse Transcription kit (QIAGEN). Quantification was performed using a LightCycler 480 Probe Master System (Roche) with primers described in Table S1. Genomic DNA from mouse tails was used as a universal standard to calculate gene copy number per ng of RNA (Yun et al., 2006).

Crhr2tm1a(KOMP)Wtsi mice on a C57BL/6 background were generated by the Knock Out Mice Program (KOMP) at the University of California Davis (Davis, CA) and Children’s Hospital Oakland Research Institute (Oakland, CA). Crhr2tm1a(KOMP)Wtsi mice were bred with mice expressing Flp recombinase to obtain Crhr2 conditional KO mice (Crhr2flox/flox mice). Crhr2flox/flox mice were backcrossed at least 10 times to wild-type C57BL/6 genetic background mice. Tamoxifen-inducible, cardiomyocyte-specific Crhr2 knockout mice (cmc-Crhr2-KOs) were generated by intercrossing the αMHC–CreERT2 line to Crhr2flox/flox mice (Takefuji et al., 2012). Cre-mediated recombination of floxed alleles was induced with an intraperitoneal injection of 1 mg tamoxifen dissolved in 100 μl Miglyol for 5 consecutive days. Vehicle-treated mice received Miglyol only, and αMHC–CreERT2+/−-Crhr2wt/wt mice were used as controls. Experiments were performed 2 wk after the end of induction.

TAC was performed under anesthesia and intubation. The chest was opened to visualize the aortic arch. The transverse aorta was then ligated between the right innominate and left common carotid arteries against a blunted 24-G needle with a 7–0 suture. The sham procedure was identical except that the aorta was not ligated.

Transthoracic echocardiography was performed on mice anesthetized with 50 mg/kg sodium pentobarbital. The left ventricular (LV) end-systolic diameter and the LV end-diastolic diameters were measured to calculate the %LV fractional shortening (%FS) in M-mode using an Acuson Sequoia C-512 (Siemens) with a 15-MHz probe. SBP was measured by tail cuff method with an automatic sphygmomanometer (BP98A; Saffron) while the mice were restrained.

Mouse plasma Ucn2 and BNP were quantified by a mouse Ucn2 ELISA kit (Yanaihara), and by a BNP Enzyme Immunoassay kit (RayBiotech Inc.), respectively, according to the manufacturer's instructions.

Histological analysis

Tissue samples were embedded in OCT compound (Sakura Finetek) and snap-frozen in liquid nitrogen. Left ventricular myocardium sections (6-µm slices) were stained with PicroSirius red with standard protocols and viewed with a BX51 microscope (Olympus). Cardiac fibrosis in 20 random images was quantified in ImageJ software.

Clinical data of human subjects

Blood samples were collected from patients diagnosed with NIDCM at Nagoya University hospital from August 2006 to November 2011. All patients showed stable disease and were hospitalized for detailed cardiac examination by laboratory analysis, echocardiography, and cardiac catheterization. NIDCM was defined as left ventricular ejection fraction (LVEF) <50% on left ventriculography and a dilated LV cavity (LV end-diastolic dimension >55 mm determined by echocardiography), in the absence of coronary heart disease, valvular heart disease, or secondary cardiac muscle disease caused by any known systemic conditions, as determined by endomyocardial biopsy. Of 52 patients, 36 (69.2%) received angiotensin-converting enzyme inhibitors or aldosterone receptor antagonists, 30 (57.7%) received β blockers, and 21 (40.4%) received mineralocorticoid receptor antagonists. Blood samples were collected from the antecubital vein of fasting patients in the morning after resting for 20 min in the supine position. Control blood samples were collected from age- and gender-matched healthy participants of a community-based cohort study without a history of metabolic, cardiovascular, or cancerous diseases. BMI was calculated as follows: BMI = weight (kg)/height (m)2. SBP and diastolic blood pressure (DBP) were measured with a sphygmomanometer, placed on the right arm of each subject with the appropriate cuff size. Plasma total cholesterol, creatinine, and glucose levels were measured by enzymatic methods. BNP (RayBiotech Inc.) and Ucn2 levels (USCN Life Science) were measured by ELISA.

RNA-sequencing

Total RNA was used to generate the cDNA library for subsequent cluster generation using the TruSeq RNA Sample Preparation kit v2 (Illumina). The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces under elevated temperature. The cleaved RNA fragments are then used for first strand cDNA synthesis with reverse transcriptase and random primers, followed by second strand cDNA synthesis. These cDNA fragments then go through an end repair process, the addition of a single “A” base, and ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. The libraries were sequenced on an Illumina HiSeq2500 platform in a paired-end 100-bp configuration.

Adapter and low-quality sequences were removed by cutadapt (v1.2.1). After quality control analysis, poly-A/T sequences were removed by PRINSEQ (v0.19.2; Schmieder and Edwards, 2011). To determine gene expression, trimmed reads were aligned to the reference human genome (GRCh37/hg19) using TopHat (v2.0.13; Trapnell et al., 2009). Mapped reads were assembled by Cufflinks (v2.2.1; Trapnell et al., 2010), and the transcripts across all samples were merged with Cuffmerge, which is part of the Cufflinks package. The fragments per kilo base per million mapped reads (FPKM) was calculated with Cuffquant, and differential expression analysis between samples was performed with Cuffdiff.

To identify candidates from the total gene pool (28,522 genes), we set the t-value cut-off at <0.01 (255 genes), and FPKM at a >1.5-fold increase compared to controls (88 genes). We then queried genes related to: CREB (29 genes), MEF2 (8 genes), or heart failure (15 genes), the CREB response element; genes harboring TGACGTCA, TGACG, or CGTCA sequences (Altarejos and Montminy, 2011); or MEF2 response elements; genes harboring a CTA(A/T)4TAG/A sequence (Akazawa and Komuro, 2003) within 3,000-bp upstream to 300-bp downstream of the transcription start site; heart failure–related genes, genes previously associated with heart failure. Six genes related to both CREB and heart failure, and one gene (Nr4a1), which was related to CREB, MEF2, and heart failure, were identified as target genes.

Study approval

The clinical study protocol was approved by the Ethics Review Board of Nagoya University School of Medicine and Jichi Medical University. Written informed consent was obtained from all study subjects. All procedures of animal care and animal use in this study were approved by the Animal Ethics Review Board of Nagoya University School of Medicine.

Statistical analysis

Data are presented as the means ± SEM in animal experiments and the means ± SD, medians (interquartile ranges), or subject numbers (%) in human subjects. All statistical analyses for animal experiments were performed in GraphPad Prism 6. Comparisons between two groups were performed with unpaired Student’s t or Chi-square tests, while those between more than two groups were done using ANOVA with Bonferroni post-hoc testing. Survival curves were analyzed with Kaplan Meyer estimators and Log-rank (Mantel-Cox) testing. Comparisons between more than two groups at different time points were performed by two-way ANOVA, followed by Bonferroni post-hoc test.

Since Ucn2 showed a skewed distribution in the human studies, the variables were log-transformed before analysis, and differences were assessed using a general linear model with parameter adjustments. Significance was defined as P < 0.05 (*, P < 0.05; **, P < 0.01).

Acknowledgments

We thank the staff from the Division of Experimental Animals, Nagoya University School of Medicine for assisting with animal experiments.

This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M. Takefuji) and by the Takeda Science Foundation (to M. Takefuji).

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